Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1984 Dec 11;12(23):8927–8938. doi: 10.1093/nar/12.23.8927

Synthesis, secretion and processing of alpha-factor-interferon fusion proteins in yeast.

A Singh, J M Lugovoy, W J Kohr, L J Perry
PMCID: PMC320429  PMID: 6083547

Abstract

A gene fusion consisting of 960 base pairs of 5'-flanking region of the yeast MF alpha 1 gene, 257 base pairs coding for alpha-factor prepro sequence, and a modified human IFN-alpha 1 gene was constructed. MAT alpha cells containing the chimeric gene synthesized and secreted active IFN-alpha 1 into the growth medium. The secreted interferon molecules contained the last 4 amino acids of alpha-factor prepro sequence and the amino acids encoded by the DNA modifications introduced at the beginning of IFN-alpha 1 gene. DNA sequences coding for these amino acids were removed by oligonucleotide-directed in vitro mutagenesis. Yeast cells transformed with expression plasmids containing the altered junction synthesized and secreted human IFN-alpha 1 with the natural NH2-terminus.

Full text

PDF
8927

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4174–4178. doi: 10.1073/pnas.73.11.4174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  3. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  6. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  7. Ciejek E., Thorner J., Geier M. Solid phase peptide synthesis of alpha-factor, a yeast mating pheromone. Biochem Biophys Res Commun. 1977 Oct 10;78(3):952–961. doi: 10.1016/0006-291x(77)90514-9. [DOI] [PubMed] [Google Scholar]
  8. Clewell D. B., Helinski D. R. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry. 1970 Oct 27;9(22):4428–4440. doi: 10.1021/bi00824a026. [DOI] [PubMed] [Google Scholar]
  9. Crea R., Horn T. Synthesis of oligonucleotides on cellulose by a phosphotriester method. Nucleic Acids Res. 1980 May 24;8(10):2331–2348. doi: 10.1093/nar/8.10.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duntze W., MacKay V., Manney T. R. Saccharomyces cerevisiae: a diffusible sex factor. Science. 1970 Jun 19;168(3938):1472–1473. doi: 10.1126/science.168.3938.1472. [DOI] [PubMed] [Google Scholar]
  11. Edman P., Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. doi: 10.1007/978-3-662-25813-2_14. [DOI] [PubMed] [Google Scholar]
  12. Emr S. D., Schekman R., Flessel M. C., Thorner J. An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7080–7084. doi: 10.1073/pnas.80.23.7080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hartley J. L., Donelson J. E. Nucleotide sequence of the yeast plasmid. Nature. 1980 Aug 28;286(5776):860–865. doi: 10.1038/286860a0. [DOI] [PubMed] [Google Scholar]
  14. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hitzeman R. A., Leung D. W., Perry L. J., Kohr W. J., Levine H. L., Goeddel D. V. Secretion of human interferons by yeast. Science. 1983 Feb 11;219(4585):620–625. doi: 10.1126/science.6186023. [DOI] [PubMed] [Google Scholar]
  16. Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Julius D., Blair L., Brake A., Sprague G., Thorner J. Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase. Cell. 1983 Mar;32(3):839–852. doi: 10.1016/0092-8674(83)90070-3. [DOI] [PubMed] [Google Scholar]
  18. Kurjan J., Herskowitz I. Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell. 1982 Oct;30(3):933–943. doi: 10.1016/0092-8674(82)90298-7. [DOI] [PubMed] [Google Scholar]
  19. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  20. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  21. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  22. Perlman D., Halvorson H. O. Distinct repressible mRNAs for cytoplasmic and secreted yeast invertase are encoded by a single gene. Cell. 1981 Aug;25(2):525–536. doi: 10.1016/0092-8674(81)90071-4. [DOI] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Singh A., Chen E. Y., Lugovoy J. M., Chang C. N., Hitzeman R. A., Seeburg P. H. Saccharomyces cerevisiae contains two discrete genes coding for the alpha-factor pheromone. Nucleic Acids Res. 1983 Jun 25;11(12):4049–4063. doi: 10.1093/nar/11.12.4049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stötzler D., Duntze W. Isolation and characterization of four related peptides exhibiting alpha factor activity from Saccharomyces cerevisiae. Eur J Biochem. 1976 May 17;65(1):257–262. doi: 10.1111/j.1432-1033.1976.tb10412.x. [DOI] [PubMed] [Google Scholar]
  26. Talmadge K., Kaufman J., Gilbert W. Bacteria mature preproinsulin to proinsulin. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3988–3992. doi: 10.1073/pnas.77.7.3988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Talmadge K., Stahl S., Gilbert W. Eukaryotic signal sequence transports insulin antigen in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3369–3373. doi: 10.1073/pnas.77.6.3369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tanaka T., Kita H., Murakami T., Narita K. Purification and amino acid sequence of mating factor from Saccharomyces cerevisiae. J Biochem. 1977 Dec;82(6):1681–1687. doi: 10.1093/oxfordjournals.jbchem.a131864. [DOI] [PubMed] [Google Scholar]
  29. Weck P. K., Apperson S., Stebbing N., Gray P. W., Leung D., Shepard H. M., Goeddel D. V. Antiviral activities of hybrids of two major human leukocyte interferons. Nucleic Acids Res. 1981 Nov 25;9(22):6153–6166. doi: 10.1093/nar/9.22.6153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES